A “mystery swine disease” appeared in the 1980's, and is present ever since in pig industry causing important economical damage worldwide (Neumann et al., 2005). The causative agent, designated porcine reproductive and respiratory syndrome virus (PRRSV), was first isolated in the Netherlands in 1991 and shortly after in the USA. It is a small enveloped positive-stranded RNA virus that is classified in the order Nidovirales, family Arteriviridae, genus Arterivirus together with equine arteritis virus, lactate dehydrogenase-elavating virus and simian hemorrhagic fever virus based on similar morphology, genomic organization, replication strategy and protein composition. In addition, they share a very narrow host tropism and a marked tropism for cells of the monocyte-macrophage lineage (Plagemann & Moennig, 1992). More specifically, in vivo PRRSV infects subpopulations of well-differentiated macrophages, with alveolar macrophages being the primary target cells, although in infected boars also testicular germ cells have been shown to allow PRRSV replication (Sur et al., 1997). In vitro, PRRSV replicates in primary cultures of alveolar macrophages and peripheral blood monocytes (PBMC), although PBMCs need treatments to improve infection (Delputte et al., 2007). Furthermore, African green monkey kidney cells and derivates thereof (Marc-145 and CL2621) have been shown to sustain PRRSV infection, although they are not from porcine origin and do not belong to the monocyte-macrophage lineage (Kim et al., 1993; Mengeling et al., 1995). Notwithstanding this very restricted cell tropism of PRRSV, the virus is able to replicate in several non-permissive cell-lines upon transfection of its viral RNA, indicating that cell tropism is determined by the presence or absence of specific receptors on the cell surface or other proteins involved in virus entry (Kreutz, 1998; Meulenberg et al., 1998).
So far, two PRRSV receptors were identified on macrophages, namely heparan sulphate (Delputte et al., 2002) and sialoadhesin (Vanderheijden et al., 2003; Wissink et al., 2003). In addition, Wissink et al. (2003) found a 150 kDa protein doublet to be involved in PRRSV infection of macrophages, however the identity of the N-glycosylated proteins is still unknown. In the current model for PRRSV infection of macrophages, PRRSV first binds to heparan sulphate most likely leading towards virus concentration. However, this first binding is rather unstable and is followed by binding to sialoadhesin and subsequent internalization (Delputte et al., 2005). Upon internalization, the virus is transported towards endosomes were a drop in pH is required for proper virus replication (Kreutz & Ackermann, 1996; Nauwynck et al., 1999). Despite this elegant research, the model is still incomplete. Transient expression of sialoadhesin in non-permissive PK-15 cells results in binding and internalization of the virus, but fusion and uncoating of the virus particles was not observed (Vanderheijden et al., 2003), indicating that other proteins are needed for virus disassembly, essential for virus replication.
PRRSV infection of Marc-145 cells makes use of a heparin-like molecule on the surface of Marc-145 cells (Jusa et al., 1997), resembling the initial step of PRRSV infection of macrophages. However, since sialoadhesin is absent from Marc-145 cells, virus entry will differ between the two cell-types. In Marc-145 cells, the intermediate filament vimentin has been described to bind to the PRRSV nucleocapsid protein and it has been suggested to interact with other cytoskeletal filaments to mediate transport of the virus in the cytosol (Kim et al., 2006). Recently, CD151 was found to interact specifically with PRRSV 3′ untranslated region (UTR) RNA (Shanmukhappa et al., 2007). CD151 was proposed to be possibly involved in fusion between the viral envelope and the endosome or to relocalize the ribonucleoprotein complexes to promote viral replication. Still, further research is needed to elucidate their precise molecular modes of action during PRRSV infection.
Recently, the scavenger receptor CD163 has been described to play a role in PRRSV infection of Marc-145 cells and to make some non-permissive cells somewhat susceptible to PRRSV upon expression (Calvert et al., 2007), where others remain unproductive upon infection, despite expression of CD163 (Calvert et al., 2007). Although the CD163 gene was originally isolated from macrophages, thus far no role for CD163 in PRRSV infection of its primary target cells has been shown. Also, the mechanism by which CD163 confers partial susceptibility of selected cell types to PRRSV infection was not elucidated.
We demonstrated that both sialoadhesin and CD163 are involved in PRRSV infection of macrophages. In addition, expression of recombinant forms of both CD163 and sialoadhesin in non-permissive cells renders all of them susceptible to PRRSV infection resulting in the production and release of infectious progeny virus. In contrast, when only CD163 is present, infection is clearly less efficient, and even absent in some cell types. In addition, viral adaptation that leads to antigenic differences in viral strains grown in cells only expressing CD163 when compared to the wild type viruses, has been reported.
Based on detailed analysis of the kinetics of PRRSV infection, both in primary macrophages and in cells expressing sialoadhesin and CD163, a role for CD163 in virus fusion and uncoating is proposed. Compared to the above mentioned systems, i.e. cells solely expressing CD163 or sialoadhesin, it has been found that the combination of CD163 and sialoadhesine expression in one cell provides permissive cells that are highly efficient to sustain viral replication, and which closely mimic the entry of the virus in the natural host, i.e. the known subpopulations of well-differentiated macrophages, in particular alveolar macrophages being the primary target cells of the virus. Such mimicry of the entry of the natural target cells will certainly reduce or avoid virus adaptation in cell culture and the associated genetic and antigenic changes that might result in viruses with altered epitopes. Such modified epitopes can have tremendous effects on the antigenicity of vaccine viruses produced on given cells, resulting in loss of induction of important neutralizing antibodies. Clearly, avoiding changes in epitopes associated with adaptation during cell culture will be beneficial for production of vaccine virus.
The results presented show that Sn and CD163 work synergistically, since co-expression of both molecules results in higher virus production compared to expression of either of the two receptors alone, and this in all cell types tested. In addition, the molecular basis of this synergistic effect was elucidated, being the receptors acting at different steps during virus entry. Sn is expressed on the surface of target cells and very efficiently captures the virus and internalizes it into the cell in endosomes. CD163 on the other does not interact with the virus on the cell surface and does not internalize the virus, but co-localizes with the virus in early endosomes, where it mediates virus uncoating, followed by release of the RNA genome in the cytoplasm. Once the genome is released in the cytoplasm, genome translation, transcription and virus replication can proceed.
The finding that CD163, which is also expressed on the cell surface, does not act on the cell surface during infection, but rather interacts with the virus in endosomes inside the cell, is quite surprising. Generally, cellular receptors act during virus attachment or internalization, or direct fusion at the cell surface. This model, in which the CD163 receptor is not active on the cell surface but acts on the virus in endosomes is surprising and explains the unanticipated and cooperative action of both Sn and CD163 during virus infection, resulting in very efficient virus infection and production of high titers of virus.
Thus, these results provide new means to generate a PRRSV permissive cell that allow for efficient viral replication, with less adaptation and accordingly solves the problems recognized in the art.